Plasmin activity assay kit

Cells were seeded at 1 × 10⁵ cells per well on a 96-well plate and adhered overnight. Cells were serum-starved overnight and then incubated with 1 µM Glu- or Lys-Plg, as specified in figure legends; Plg concentration was determined experimentally after testing doses from 1–10 µM (data not shown), and the lowest dose was chosen to mimic normal serum Plg concentrations [36 (link)]. A plasmin activity assay was then run according to the manufacturer’s specification (Plasmin Activity Assay Kit, Abcam, Cambridge, UK). Briefly, after cell incubation with test samples for 1–12 h, as indicated in figure legends, 50 µL of plasmin substrate was added to each well. Substrate cleavage was measured by fluorescence detection at Ex/Em 360/450 nm in kinetic mode (read every 2.5 min for 22 min) on a Synergy HTX Multi-Mode Microplate Reader. To measure activity in the supernatant, experiments were set up as above, then supernatants collected and moved to clean assay wells following a 1 h incubation with Plg. Supernatants were replaced on cells with serum-free media. Pla substrate was then added to cells and supernatants separately, and activity assessed as above.

The plasmin activity was assayed in cell supernatants collected from HUVEC and HNEpC (seeded in a 24-well plastic plate at 1.5 × 10⁵ cells per well) as reported in [16 (link)]. Beside the solutions of interest, the mesoglycan was also used at 300 µg/mL for 24 h as positive control and the apotrinin (Sigma-Aldrich; St. Louis, MO, USA), as a serine protease inhibitor, administered at 30 µM, 10 min before reading. The samples were analyzed through the Plasmin Activity Assay kit (Abcam, Cambridge, UK) following the manufacturer’s instructions and adapted to cell media. Particularly, the standard curve was created, starting from a plasmin concentration of 0 ng/well (in 50 µL of standard volume/well) to 75 ng/well. A total of 50 µL of each experimental point was used for reading in triplicate. A total of 50 µL of reaction mix (plasmin assay buffer + plasmin substrate) was added to standard and sample wells. The output fluorescence was evaluated as Ex/Em = 360/450 nm at the EnSight Multimode Plate Reader (PerkinElmer; Waltham, MA, USA) in a kinetic mode (a reading each 2 min from 10 to 20 min of plate incubation at 37 °C). The amount of plasmin was calculated, as previously reported.